A key event in normal cells and atherosclerotic lesional macrophages is the trafficking of lipoprotein- cholesterol (LP-Chol), i.e., LP-Chol uptake, processing, and transport from late endosomes to the plasma membrane and endoplasmic reticulum (ER). However, little is known about the molecular machinery that facilitates these processes. The overall objective of this proposal is to identify genes and proteins that mediate LP-Chol trafficking and to elucidate the mechanism of these proteins. The overall hypothesis is that trafficking of LP-Chol involves vesicular and non-vesicular mechanisms mediated by a number of heretofore undiscovered proteins. In this context, the objectiveswill be met by using innovative, interdisciplinary approaches to carry out the following three Aims: (I) A photocholesterol-based proteomics strategy is being used to identify proteins that interact with LP-Chol and facilitate its trafficking. In our first series of experiments, which has focused on early events in the trafficking itinerary, we identified a protein called PDI- related protein 5 (P5). Based on preliminary data, we hypothesize that P5, either directly or through interaction with another protein, facilitates LP-Chol uptake, processing, or transport to the ER. This Aim will further explore the site of action and mechanisms of P5 as well as screen for additional proteins that are involved in the later stages of LP-Chol trafficking; (II) a highly efficient and comprehensive shRNA library is being used in a candidate gene approach and in a high-throughput fluorescence microscopy screen of LP-Chol trafficking. The candidate approach has identified StarDS as a protein that appears to be involved in the transportor distribution of late endosomal cholesterol. We will study the function and mechanism of this protein as well as other proteins identified in both the candidate approach and in the high-throughput screen; and (III) In-depth examination of the trafficking of LP-derived dehydroergosterol, a fluorescent cholesterol homologue, will be used to test hypotheses related to LP-Chol trafficking pathways involving endocytosis, late endosomes, the plasma membrane, and the ER, particularly under situations in which the proteins discovered in Aims I and II, including P5 and StarDS, are disabled by RNAi. After achieving these goals, we hope to have a much deeper understanding of the molecular mechanisms of LP-Chol trafficking, particularly in the context of atherosclerotic lesional macrophages that internalize atherogenic lipoproteins.